Brazilian nut consumption by healthy volunteers improves inflammatory parameters.

OBJECTIVE: The aim of this study was to investigate the effect of a single dose of Brazil nuts on the inflammatory markers of healthy individuals. METHOD: A randomized crossover study was conducted with 10 healthy individuals (mean age 24.7 ± 3.4 y). Each individual was tested four times regarding intake of different portions of Brazil nuts: 0, 5, 20 and 50 g. At each testing period, peripheral blood was collected before and at 1, 3, 6, 9, 24, and 48 h after intake of nuts, as well as at 5 and 30 d after intake of various Brazil nut portions. Blood samples were tested for high-sensitivity to C-reactive protein, interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, aspartate and alanine aminotransferases, albumin, total protein, alkaline phosphatase, gamma-glutamyltransferase, urea, and creatinine. RESULTS: Consumption of nuts did not affect biochemical parameters for liver and kidney function, indicating absence of hepatic and renal toxicity. A single intake of Brazil nuts (20 or 50 g) caused a significant decrease in serum IL-1, IL-6, TNF-α, and IFN-γ levels (P < 0.05), whereas serum levels of IL-10 were significantly increased (P < 0.05). CONCLUSION: The results indicate a long-term decrease in inflammatory markers after a single intake of large portions of Brazil nuts in healthy volunteers. Therefore, the long-term effect of regular Brazil nut consumption on inflammatory markers should be better investigated.

Physiological (antioxidant) responses of estuarine fishes to variability in dissolved oxygen.

Cycles of dissolved oxygen (DO) in estuaries can range from anoxia to various levels of supersaturation (200-300%) over short time periods. Aerobic metabolism causes formation of damaging reactive oxygen species (ROS), a process exacerbated by high or low DO. Fish can generate physiological defenses (e.g. antioxidant enzymes) against ROS, however, there are little data tying this to environmental conditions. We investigated physiological defenses generated by estuarine fishes in response to high DO and various DO cycles. We hypothesized that chemical defenses and/or oxidative damage are related to patterns of DO supersaturation. Specific activities of antioxidants in fish tissues should be positively correlated with increasing levels of DO, if high DO levels are physiologically stressful. We caged common benthic fishes (longjaw mudsucker, Gillichthys mirabilis, and staghorn sculpin, Leptocottus armatus, in CA and spot, Leiostomus xanthurus and pinfish, Lagodon rhomboides, in NC) during summer 1998 in two estuarine sites in southern North Carolina and two in central California. At each site a water quality meter measured bottom DO, salinity, temperature, depth, pH and turbidity at 30 min intervals throughout the study. These sites exhibited a wide variety of dissolved oxygen patterns. After 2 weeks in the cages, fish gills and livers were analyzed for antioxidant enzymes (glutathione peroxidase, catalase and superoxide dismutase) and the metabolite glutathione. All fish exhibited antioxidant enzyme activity. There was a significant site-dependent effect on all enzyme activities at the NC sites, with the most activity at the site with the highest DO cycling and the most DO supersaturation. There was a trend towards higher enzyme activities under high DO levels at the CA sites.

The antioxidants of legume nodule mitochondria.

The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation. They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites. We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L. cv. Contender x Rhizobium leguminosarum bv. phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix. This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase. Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids. Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts. We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane. The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol. In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase.

Stress-induced legume root nodule senescence. Physiological, biochemical, and structural alterations.

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2-4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by gamma-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.

Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems.

Ascorbate and ascorbate peroxidase are important antioxidants that are abundant in N2-fixing legume root nodules. Antioxidants are especially critical in root nodules because leghemoglobin, which is present at high concentrations in nodules, is prone to autoxidation and production of activated oxygen species such as O2.- and H2O2. The merits of ascorbate and ascorbate peroxidase for maintaining conditions favorable for N2 fixation were examined in two model systems containing oxygen-binding proteins (purified myoglobin or leghemoglobin) and N2-fixing microorganisms (free-living Azorhizobium or bacteroids of Bradyrhizobium japonicum) in sealed vials. The inclusion of ascorbate alone to these systems led to enhanced oxygenation of hemeproteins, as well as to increases in nitrogenase (acetylene reduction) activity. The inclusion of both ascorbate and ascorbate peroxidase resulted in even greater positive responses, including increases of up to 4.5-fold in nitrogenase activity. In contrast, superoxide dismutase did not provide beneficial antioxidant action and catalase alone provided only very marginal benefit. Optimal concentrations were 2 mM for ascorbate and 200 micrograms/ml for ascorbate peroxidase. These concentrations are similar to those found in intact soybean nodules. These results support the conclusion that ascorbate and ascorbate peroxidase are beneficial for maintaining conditions favorable for N2 fixation in nodules.

Class I heme peroxidases: characterization of soybean ascorbate peroxidase.

An efficient expression system [D. A. Dalton et al. Arch. Biochem. Biophys. 328, 1-8, 1996) for soybean nodule ascorbate peroxidase (APX) has, for the first time, been used to generate enzyme in large enough quantities for detailed biophysical analysis. The recombinant APX has been characterized by electronic absorption, EPR, NMR and circular dichroism spectroscopies, and by electrochemistry. Electronic, EPR, and NMR spectra are consistent with a high-spin ferric resting state for the enzyme at 298 K. Low-temperature EPR (7 K) and electronic absorption (77 K) experiments indicate formation of a low-spin heme derivative at these temperatures. The midpoint reduction potential for the Fe(III)/Fe(II) redox couple, determined by spectroelectrochemistry, is -159 +/- 2 mV vs SHE (pH 7.0, 25.0 degrees C, mu = 0.10 M). Circular dichroism spectra of pea and soybean APXs are very similar, indicating common structural features for the two enzymes. The melting temperature of soybean APX, as monitored by circular dichroism spectroscopy, is 49 degrees C. These results represent the first detailed spectroscopic and electrochemical analysis of soybean ascorbate peroxidase and are discussed in the broader context of other class I peroxidases.

Antioxidant defenses in the peripheral cell layers of legume root nodules.

Ascorbate peroxidase (AP) is a key enzyme that scavenges potentially harmful H2O2 and thus prevents oxidative damage in plants, especially in N2-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago sativa) root nodules contains elevated levels of AP protein, as well as the corresponding mRNA transcript and substrate (ascorbate). Enhanced AP protein levels were also found in cells immediately peripheral to the infected region of soybean (Glycine max), pea (Pisum sativum), clover (Trifolium pratense), and common bean (Phaseolus vulgaris) nodules. Regeneration of ascorbate was achieved by (homo)glutathione and associated enzymes of the ascorbate-glutathione pathway, which were present at high levels. The presence of high levels of antioxidants suggests that respiratory consumption of O2 in the endodermis or nodule parenchyma may be an essential component of the O2-diffusion barrier that regulates the entry of O2 into the central region of nodules and ensures optimal functioning of nitrogenase.

Heterologous expression and characterization of soybean cytosolic ascorbate peroxidase.

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules.

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.

Purification and characterization of monodehydroascorbate reductase from soybean root nodules.

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.

Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules.

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of O(2). The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high pO(2). Nodule contents of ascorbate and reduced glutathione were also greater in the high pO(2) treatments. Treatment of nodulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N(2) fixation and defenses against activated forms of oxygen.

Nickel as a micronutrient element for plants.

The detrimental effects of excessive Ni on plant growth have been well known for many years. More recent evidence indicates that Ni is required in small amounts for normal plant growth and development. Ni is an essential component of urease in plants and microorganisms. A deficiency of Ni in plants is reported to result in necrotic lesions in leaves in response to toxic accumulations of urea. Urease plays an essential role in mobilization of nitrogenous compounds in plants, a process that is especially important during seed germination and fruit formation when protein reserves are degraded into amino acids. Arginine, an abundant amino acid in plants, when degraded produces urea as a product and urease is needed for urea utilization. Theories of urea formation during allantoin degradation in Glycine max have been recently refuted. In G. max ureides apparently are metabolized via an amidohydrolase reaction with subsequent degradation of ureidoglycine, yielding glyoxylate, NH+4 and CO2. No evidence is available for the formation of urea in this pathway. Nitrogen-fixing symbionts, such as Rhizobium and Bradyrhizobium, contain two known Ni enzymes: urease and hydrogenase. Optimum growth of nodulated legumes and actinorhizal plants may depend on an adequate supply of Ni to meet the requirements of the Ni-requiring enzymes in host plants and endophytes. The seeds of severely Ni-deficient Hordeum are completely inviable, thus providing conclusive evidence for the essentiality of Ni for this species. The evidence indicates that Ni must be added to the list of micronutrient elements generally required by plants.

Purification, properties, and distribution of ascorbate peroxidase in legume root nodules.

All aerobic biological systems, including N(2)-fixing root nodules, are subject to O(2) toxicity that results from the formation of reactive intermediates such as H(2)O(2) and free radicals of O(2). H(2)O(2) may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN(3), CO, and C(2)H(2) were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of (1/2) V(max) was 70 micromolar for ascorbate and 3 micromolar for H(2)O(2). The high affinity of ascorbate peroxidase for H(2)O(2) indicates that this enzyme, rather than catalase, is responsible for most H(2)O(2) removal outside of peroxisomes in root nodules.

Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules.

The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (Glycine max). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H(2)O(2) in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.